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Research has been conducted on the Drosophila melanogaster homeodomain dynamic behavior. The role of the residues in the C-terminal region of the DNA recognition helix has been investigated via the (super)15 N relaxation studies of the wild type and H52R/T56W mutant of the vnd/NK-2 homeodomain in the free and DNA-bound state.

The conformational stability of the wild-type homeodomain of the vnd/NK-2 gene of Drosophila melanogaster is compared to the same homeodomain with one and two amino acid replacements in the DNA-binding region. Results indicate that thermodynamic parameters reflect solvent and DNA rearrangements as well as structural changes caused by DNA binding.

The selectively deuterated isoleucines I3, I13, I16 and I20 and solid-state 2H NMR were used in studying the behavior of the cytolytic peptide fragment 828-848 (P828) from the carboxy-terminus of the envelope glycoprotein gp41 of HIV-1. Results indicate a significant decrease in the lipid chain order toward the bilayer center, but not in the lipid glycerol, due to the incorporation of peptides. This findings suggest that the peptide backbone has been penetrated into the membrane/water interface region which reduces the lateral packing density and reduces order in the hydrophobic core.

The structural and DNA binding behavior is described for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue alanine to threonine replacement in position 35 of the homeodomain. Multidimensional nuclear magnetic resonance, circular dichroism, and electrophoretic gel retardation assays were carried out on recombinant 80-aa residue proteins that encompass the wild-type and mutant homeodomains. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded conformation free in solution at temperatures down to -5 [degrees] C in contrast to the behavior of the corresponding wild-type vnd/NK-2 homeodomain, which is folded into a functional three-dimensional structure below 25 [degrees] C. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain. Although the three-dimensional structure of the mutant A35T vnd/NK-2 in the DNA bound state shows characteristic helix-turn-helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the A35T mutant this amide proton resonance is shifted upfield by 1.27 ppm relative to the resonance frequency for the wild-type analog, thereby indicating a significant alteration of this i,i-5 hydrogen bond.

The interactions between the helix III and the DNA major groove was established through analyzing the intermolecular protein-DNA distances through spectroscopy and molecular modeling computations. Data indicated that the specificity is determined by amino acid residue 54 while transduction from position L7 to the DNA phosphate backbone relative to guanosine/cytosine base pair preference demonstrated the binding specificity for the N-terminal region.

Two-dimensional and three-dimensional NMR spectroscopic studies of the secondary structure of the homeodomain encoded by the NK-2 gene from Drosophila melanogaster reveal the presence of three helical units with homeodomain amino acid residues 10-22, 28-38 and 45-52 in the absence of DNA. The third helix undergoes a length extension to 19 amino acids when the homeodomain is bound to the DNA. The thermal stability of the protein is enhanced on complexing NK-2 with the duplex DNA.

Electrospray mass spectrometry was used to confirm the identity and purity of the unlabeled and isotopically labeled vnd/NK2-A35T homeodomain protein prepared for NMR experiments. A series of events involving adduction by urea, carbamoylation of amino acid groups, and the presence of nickel ion resulted in protein masses shifted from their expected values, thus emphasizing that observed discrepancies in mass may have sources other than an error in amino acid sequence. Procedures used to detect and remedy these problems are described.

Thermodynamics parameters are reported for Nkx2.5(C56S) binding specific, 18 bp DNA at pH 7.4 in the presence of 100 mM NaCl and the temperature dependence of DNA binding affinity as well as the enthalpic and entropic contributions from 10 to 37 degree Celsius. Thermodynamic parameters differ for Nkx2.5 and vnd/NK-2 homeodomains binding specific DNA.

A multidimensional heteronuclear NMR study to determine the structure of Aux-C20 is presented. The result proposed the J-domain protein substrate binding domains and their relationship to their respective J-domains appear to be individually adapted to their specific substrates

The determination of the three dimensional solution structure obtained by NMR of the A35T mutant vnd/NK-2 homeodomain bound to the vnd/NK-2 consensus 16 bp DNA sequence is discussed. A detailed comparison of the structures of the mutant A35T and wild type vnd/NK-2 homeodomains bound to cognate DNA is presented.